Asthma prevalence is greater in boys than girls, but around puberty there is a shift in asthma prevalence, and at mid-life women are about two times more likely than men to have asthma. This suggests a role for sex hormones in asthma pathogenesis; however the mechanisms remain unknown. Group 2 innate lymphoid cells (ILC2) are important in driving the initial phase of allergic airway inflammation that is associated with asthma. ILC2 are activated by IL-33, TSLP, and IL-25 which are upregulated in response to airway allergens, including Alternaria alternata. Upon stimulation, ILC2 have increased expression of the transcription factors GATA3 and ROR? and produce IL-5 and IL-13. IL-5 and IL-13 increase the infiltration of eosinophils, airway hyperresponsiveness (AHR), and mucus production, all hallmarks of asthma. Our preliminary data showed IL- 33-stimulated ILC2 from female mice had significantly increased IL-5 and IL-13 protein expression compared to ILC2 from male mice. Therefore, we hypothesize that the ovarian hormones, 17beta-estradiol (17?-E2) and progesterone (P4), increase ILC2-induced airway inflammation. In Aim 1 we will determine the mechanisms by which sex hormones increase IL-5 and IL-13 production from ILC2. Sham-operated female and male mice, ovariectomized female mice, and orchidectomized male mice will be administered pellets containing vehicle, 17?-E2, P4, and/or testosterone. ILCs will be harvested from the bone marrow and lungs of these mice and stimulated with IL-33 ex vivo. IL-5 and IL-13 protein expression, ROR? and GATA3 mRNA expression, and surface expression of ST2, a component of the IL-33 receptor, will then be determined in ILC2. In Aim 2, we will determine the role of ovarian hormones on Alternaria extract (Alt Ex)-induced innate immune-mediated airway inflammation. WT BALB/c female, male, ovariectomized, and orchidectomized mice will be challenged with Alt Ex for 4 days to initiate an innate immune response. We will then determine ILC2 cytokine expression, airway inflammation, AHR, and mucus production. This proposal will delineate a mechanism(s) by which sex hormones regulate ILC2, and it may identify potential therapeutic targets for patients, in particular women, with asthma.